International Immunology, Vol. 20, No. 9, pp. 1211–1218
doi:10.1093/intimm/dxn079
ª The Japanese Society for Immunology. 2008. All rights reserved.
For permissions, please e-mail: [email protected]
Manipulation of immune system via immortal bone
marrow stem cells
Christiane Ruedl, Hanif Javanmard Khameneh and Klaus Karjalainen
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
Keywords: hematopoietic stem cells, manipulation of immune system, NUP98-HOXB4
Abstract
Extensive amplification of hematopoietic stem cells (HSCs) and their multipotent primitive
progenitors (MPPs) in culture would greatly benefit not only clinical transplantation but also provide
a potential tool to manipulate all cellular lineages derived from these cells for gene therapy and
experimental purposes. Here, we demonstrate that mouse bone marrow cultures containing cells
engineered to over-express NUP98–HOXB4 fusion protein support self-renewal of physiologically
normal HSC and MPP for several weeks leading practically to their unlimited expansion. This allows
time consuming and cumulative in vitro experimental manipulations without sacrificing their ability to
differentiate in vivo or in vitro to any hematopoietic lineage.
Introduction
Adult bone marrow (BM) houses a tiny pool of hematopoietic stem cells (HSCs) that have the ability to maintain not
only themselves but also all the rest of highly turning over
blood lineages throughout the mammalian life (1, 2).
Hence, the ability to sustain HSC in tissue culture would allow serial introduction of gain or loss of function mutations
efficiently in hematopoietic system. However, our failure to
expand HSC in culture has hampered the use of this approach. In fact, BM suspension cultures lose rapidly their
HSC content despite vigorous growth of progenitors and
more differentiated cells at least for 3 weeks even in optimal cytokine milieu (3, 4). Therefore, the phenomenon of
stem cell exhaustion or senescence may set the limits that
make it impossible even in principle to expand HSC in culture for longer periods (5–7).
Mouse HSC do expand in vivo (8, 9), at least up to 8000fold, as shown by Iscove and Nawa (9) through serial transplantation experiments that assessed carefully the input
and output contents of HSC in each transfer generation.
Recently also in vitro approaches have been improved and
refined culture conditions with new growth factors can now
support up to 30-fold expansion of mouse HSC ex vivo
(10). However, since it is not clear to what extent external
culture conditions can be improved, alternative but not mutually exclusive efforts to change the intrinsic properties of
HSC have been taken. Seminal experiments in this respect
by Humphries, Savageau and their colleagues have shown
that ectopic expression of HOXB4 transcription factor in
BM cells support the survival and expansion of HSC in vivo
Correspondence to: Christiane Ruedl; E-mail: [email protected]
Transmitting editor: M. Reth
and importantly also in vitro (11–13). By rigorously monitoring the HSC content in their cultures of HOXB4-transduced
BM cells, they found that HSC could be expanded up
to 41-fold in the 2-week liquid cultures (13). HOXB4
belongs to a large family of HOX transcription factors that
are crucial for the basic developmental processes in addition to their role in maintenance of different stem cell
compartments.
Capitalizing on the findings of Humphries, Savageau and
their colleagues, we have established long-term murine BM
cultures of HOXB4-transduced cells (HOX cells) and monitored their stem cell content to find out how extensively
genetically modified HSC and their multipotent primitive progenitors (MPPs) can be expanded in culture for experimental
purposes. In addition and for comparison, we established
BM cultures transduced with constructs encoding for
Nucleoporin 98 (NUP)–HOXB4 (NUP cells) fusion protein
again following the lead of Humphries et al. (14) who
showed that ectopic expression of similar fusions promoted
in vivo even more robust expansion and survival of HSC.
Many HOX family members when over-expressed ectopically in BM cells induce leukemias when transplanted in vivo
(15–17) as already suggested by their dysregulated expression in hematological malignancies (18–20). Similarly, many
NUP98 HOX fusion products detected especially in acute myelogenous leukemias are leukemogenic when ectopically
expressed in BM cells (14, 21, 22). Importantly, however,
over-expression of HOXB4 and NUP98-HOXB4 genes seems
not to induce leukemias experimentally in mice keeping with
Received 25 April 2008, accepted 17 June 2008
Advance Access publication 21 July 2008
1212 Immortalized bone marrow stem cells
Fig. 1. Surface phenotype of HOXB4 and NUP98-HOXB4-transduced BM cells as well as that of HSC within transduced cells. (A) Representative
FACS analysis of cells from 4-week-old cultures is shown. Cultured cells were characterized for the expression of Sca-1, c-Kit, FcReI, CD27, Gr-1
and Mac-1 (B and C) Cells from a 4-week-old HOX culture were sorted into four fractions according to their Sca-1 and FcReI expression levels as
indicated (B). Cells from each fraction (2 3 105, Ly5.1) were injected together with normal BM cells (2 3 105, Ly5.2) into lethally irradiated
recipients (Ly5.2). NUP cell contribution to the sustained and multilineage reconstitution was then measured 4 months later in blood (C). Robust
HSC activity was only detectable in fraction which corresponds to LSK fraction. Multilineage repopulation was determined as in Fig. 3 (data not
shown). One typical recipient out of four is shown.
the fact that fusion products of NUP98-HOXB4 have not been
detected in leukemias (14, 22). Interestingly, it is not clear
mechanistically why the component of the nucleopore complex has so dramatic effect in hematopoiesis.
Here, we report that cultures containing HOXB4 and especially NUP98-HOXB4-transduced BM cells sustain physiologically normal HSC and their MPPs for long periods
leading practically to their unlimited expansion and hence
providing us convenient targets for the experimental manipulation of hematopoietic lineages.
and HOXB4 portion from MSCV-HOXB4-IRES-GFP. The fusion
break point was that described by Pineault et al. (14). The fusion product was then cloned into pMYc-IP retroviral vector
(24) containing IRES-puromycin resistance gene to generate
pMYc-NUP98-HOXB4-IP. In both of these final constructs, the
expression of HOXB4 and NUP98-HOXB4 was driven from
the retroviral long terminal repeat. Similarly, coding regions of
ovalbumin (OVA), programmed death-1 ligands (PD-L) and
2 were amplified by PCR and cloned in pMYc-IG vector (24).
All constructs were verified by DNA sequencing.
Methods
Transduction of mouse BM cells
Retroviral vectors
Portion encoding for human HOXB4 was transferred from
MSCV-HOXB4-IRES-GFP [gift from Sauvageau (12)] to LXSP
retroviral vector to create LHOXB4SP. LXSP is a derivative of
LXSN (23) in which puromycin-resistant gene was introduced
in the place of neomycin gene. NUP98–HOXB4 fusion gene
was created by amplifying NUP98 portion from plasmid
pcDNA3.1-3 3 HA-NUP98-HOXA9 [gift from Nakamura (21)]
Protocol of Antonchuk et al. (13) was followed with minor modifications; retrovirus-containing supernatants were generated
by transfection of Ecotropic Phoenix packaging cells instead
of GP + E86 producer line and then used to spinoculate twice
(days 2 and 3) BM cells, 3 3 105 per well, in 24-well plates.
Transduced cells were selected for puromycin resistance
(2 lg ml 1) 2 days after the last spin infection. BM and transduced cells were cultured in IMDM supplemented with 2%
FCS, 0.03% Primatone (Quest, Naarden, The Netherlands),
Immortalized bone marrow stem cells
1213
Fig. 2. Measurement of the HSC content of the cultures of the HOXB4 and NUP98-HOXB4-transduced BM cells. Level of chimerism in blood of
lethally irradiated hosts (Ly5.2) was monitored 16 weeks after transplantation. Indicated numbers of HOX and NUP cells (Ly5.1) from 25-day
(upper panel), from 60-day (middle panel) and from 74-day-old cultures (lower panel) were co-injected with 2 3 105 competitor BM cells (Ly5.2).
In each case, two independent recipients are shown. Multilineage reconstitution was analyzed as in Fig. 3.
mIL-3, mIL-6 and mSCF. Supernatants from transfected X63
plasmacytoma lines provided the sources of IL-3 and IL-6
(25). Stem cell factor (SCF) was produced in bacteria as
NH2-terminal 6–HIS fusion protein. IL-3- and SCF-dependent
HOXB4 lines were used to determine the factor concentrations for optimal growth. IL-6 was supplemented as 1:50 dilution of the original X63 supernatant in the absence of
appropriate bioassay representing most likely great excess.
Mice
C57BL/6 (B6) congenic Ly5.1 and Ly5.2 mice (gift from
Rolink) of 6–10 weeks were used. All mice were bred and
maintained in the animal facility of the Nanyang Technological
University under specific pathogen-free conditions. All animal
experiments were carried out within institutional guidelines.
For transplantation, the recipients B6-Ly5.2 were lethally
irradiated with 1000 rad in two doses separated by 4 h.
Twelve to twenty-four hours later, constant numbers of
B6-Ly5.2 BM competitor cells (2 3 105) with indicated num-
bers of cultured B6-Ly5.1 cells were injected (200 ll in PBS)
in tail vein. OT-I and OT-II mice were purchased from The
Jackson Laboratories.
Antibodies and flow cytometry
FITC-, PE- and APC-labeled antibodies (Ly5.1, Sca-1, c-Kit,
FcReI, CD27, Mac-1, CD11c, CD3, B220, Gr-1, PD-L1 and 2)
were all purchased from eBiosciences (San Diego, CA,
USA), Cell suspensions of blood, spleen, thymus and BM
and of HOXB4 or NUP cultures were stained in ice-cold
PBS supplemented with 2% FCS. Data were collected on
FACSCalibur (Becton Dickinson) and analyzed with FlowJo
software (Treestar, Ashland, OR, USA). All cell sortings were
performed by FACSaria (Becton Dickinson).
In vitro cultures for derivation of dendritic cells and antigen
presentation assay
Cultured cells were grown in the presence of granulocyte
macrophage colony-stimulating factor (GM-CSF) (1:50
1214 Immortalized bone marrow stem cells
Fig. 3. Cultured cells support sustained multilineage reconstitution. Representative example of the analysis of myelolymphoid reconstitution is
shown. Lethally irradiated recipient (Ly5.2) injected with 2 3 105 NUP cells (Ly5.1) from 25-day-old cultures together with 2 3 105 competitor BM
cells (Ly5.2) was analyzed 16 weeks after transplantation. B, T and myeloid cell compartments and main thymocyte subsets derived from
cultured cells were analyzed in blood, spleen and thymus, respectively. Overall level of chimerism in each organ is shown above. To score
monocytes and granulocytes, the gate was extended to include larger and more granular cells.
Fig. 4. Cultured HOX cells can be serially transplanted. Upper part, the general design of the experiment is shown. Lethally irradiated primary
recipients (Ly5.2) were transplanted with 105 HOX cells (Ly5.1) from a 3-week-old culture (i.e. ;106-fold expansion). Four months later, BM cells
from primary recipients were cultured further for 1 week and then injected (106) to lethally irradiated secondary recipients (Ly5.2). Again 4 months
later, BM cells (106) from secondary recipients were transferred directly to lethally irradiated tertiary recipients (Ly5.2) and finally 4 months later
the tertiary BM cells were isolated for analysis. Lower part shows the level of chimerism obtained from cultured cells in each transfer generation in
BM. Three mice in each generation were analyzed with similar results. Multilineage reconstitution was determined as in Fig. 3 (data not shown).
Immortalized bone marrow stem cells
1215
Fig. 5. Cultured NUP cells can be differentiated in vitro to functional DC. Cells from a 4-week-old NUP culture were differentiated in vitro in the
presence of either FLT3L (upper panel) or GM-CSF (lower panel) for 7 or 10 days, respectively. Cells were then stained with antibodies against
CD11c versus MHC class II, CD11b (for myeloid-related DCs) and B220 (for plasmacytoid DCs) and analyzed by FACS (A). DC derived from
NUP cells (GM-CSF cultures shown) stimulates antigen-specific T cells (OT-1) as efficiently as those derived from normal BM (B). DCs were
pulsed with OVA peptide and different numbers were then placed in standard T cell stimulation assay.
dilution of transfected X63 plasmacytoma) (26) or FLT3L
(10 ng ml 1) for 7 or 10 days, respectively. Dendritic cell (DC)
generation was monitored by double staining using APClabeled anti-CD11c combined with PE-labeled anti-MHC
class II, anti-CD11b (for myeloid-related DCs) or anti-B220
(for plasmacytoid DCs) using a FACSCalibur.
GM-SCF-derived DCs from NUP98-HOXB4 lines as well as
from normal BM cells were in vitro pulsed with 100 lg ml 1
of OVA and co-cultured for 3 days with OVA-specific CD4+
T cells from OT-II transgenic mice. T cell proliferation was
assessed by measurement of [3H]thymidine incorporation.
Results
Generation of ‘immortalized’ lines
The protocol established by Antonchuk et al. (13) was followed to transduce BM cells from 5-Fluoracil-treated mice
with standard retrovirus vectors coding either for HOXB4 or
NUP98-HOXB4 proteins. Transduced cells were then maintained in six-well plates in SCF, IL-3 and IL-6 containing media at cell concentrations between 105 5 3 105 by feeding
and splitting the cultures in five or six (in a new well) every
2–3 days—cells divided every 20–22 h (data not shown)
maintaining robustly that rate regardless of the age of the
culture (1 week to 3 months).
Phenotype of transduced cells
Cultured cells were monitored weekly for their surface phenotype in order to reveal potential telltale signs of culture
‘evolution’. Snapshots of 4 week cultures representing typical phenotype are shown (Fig. 1A). At this time, cells in
normal cultures (untransduced) had died off or only some
slow-growing mast cell precursors were present. Generally,
NUP cells keep their primitive phenotype quite tightly being
1216 Immortalized bone marrow stem cells
mostly Lin Sca-1+c-Kit+ (LSK) phenotype (1). HOX cells
show a clear predilection to mast cell lineage in these conditions (FcReI+, either Sca-1+/c-Kit+ or Sca-1 /c-Kit ) although
the abundance of these sub-populations fluctuates somewhat in weekly samplings. Cultures are devoid of erythroid
(Ter119+) and B (B220+) lymphoid precursors as well as
T lymphocytes (CD3+) (data not shown) but contain some myeloid cells (Gr1+/MAC-1+). These phenotypes were relatively
stable through at least 3 months of culture (duration of the experiment) during which cultures kept robustly expanding in
growth factor-dependent way (data not shown) and no further
attempts to optimize culture conditions were made.
Transduced HSC have a normal cell surface phenotype
It is important to note that the phenotype does not necessarily have to represent some physiological BM sub-population
since over-expression of HOXB4 or NUP98-HOXB4 can directly or indirectly influence the pattern of surface expression. However, like in the case of normal BM cells, all
detectable HSC reside in LSK fraction of the HOX cells. This
was shown by testing the presence of HSC in different
sorted populations from 4-week-old HOX cultures (Fig. 1B).
Only LSK cells (Sca-1+/c-Kit+/FcReI ) gave sustained multilineage reconstitution in the presence of competitor cells in
standard in vivo HSC assays (Fig. 1C and see below). This
also suggests that ectopic expression of HOX does not increase potential ‘plasticity’ of BM cells in such an extent that
they would be able to dedifferentiate from more mature cells
back to stem cell-like state.
Transduced cultures sustain their HSC content
Since our cultures seem to support phenotypically MPPs that
could be generated continuously from HSC, we wanted to
quantify the actual HSC content in our cultures. To do this,
we exploited the competitive BM reconstitution assay
whereby titrated numbers of cultured cells from B6-Ly5.1
mice were forced to compete against the constant number
(2 3 105) of normal, freshly isolated congenic B6-Ly5.2 BM
cells in lethally irradiated recipients (B6-Ly5.2) (27). The
level of reconstitution in the blood myeloid and lymphoid
compartments were analyzed earliest 16 weeks after transplantation to avoid scoring short-term HSC (26, 28) and at
the end point of a given experiment also lymphoid organs
like thymus was analyzed in order to get further evidence
for active and continuous lymphopoiesis. Small sizes of our
experimental groups 2–3 mice in each prevented us to perform proper in vivo fluctuation analysis. Therefore, the last
experimental dose giving the robust (>3%) and sustained
multilineage reconstitution in every mouse within the group
was considered to contain at least one HSC i.e. our measurement were estimates (Figs 2 and 3).
Sampling of the cultures for their HSC content were
started at day 25 when control i.e. untransduced BM cultures had stopped their vigorous growth and accumulated
only adherent cells and some slowly growing mast cell precursors. At this time point, transduced cells had expanded
;225-fold (;3 3 107) and hence our surprise that HSC
could be measured in high frequency (Figs 2 and 3). NUP
cultures contained still at least 1 HSC/2 3 104 cultured cells
Fig. 6. Cultured NUP cells can be cumulatively modified without
sacrificing their potential to differentiate in vivo. Experimental outline is
shown above (A). Cells from a 3-week-old NUP cultures were first
transduced with OVA-IRES-GFP-containing retrovirus. After cell
sorting, green cells were further transduced with PD-L1-IRES-GFP
particles and after second cell sort the selected (PD-L1+) cells were
finally transduced and selected for PD-L2 expression. Expression
levels of PD-L1 and 2 on NUP cells from the final step are shown. The
staining of untransduced cells is shown as empty histograms (B, left
panel). Triply transduced and selected NUP cells were injected in the
lethally irradiated congenic recipients (106 per recipient). Three
weeks later, DCs were isolated from spleens of the recipients and
analyzed for PD-L1 and 2 surface expression. Freshly isolated
normal, untransduced, DC served as controls (empty histograms)
(B, right panel). More than 90% of the DCs were derived from
modified cells (data not shown). One typical example out of six
recipients is shown.
representing only ;10-fold decrease in the frequency compared with that of initial cultures of 5-FU-treated BM cells
measured to be 1/2000 (27) (Fig. 2, upper panel). HOX cultures maintained HSC less well but still in easily detectable
frequencies of 1/100 of the starting culture. Hence, in NUP
cultures, HSC had expanded more than million fold (;3 3
106) while in HOX cultures they did so 10-fold less efficiently
correlating nicely with the phenotypic analysis (Fig. 1). Similar results were obtained with cultures initiated and propagated independently for 25 days, demonstrating that these
cultures robustly and reproducibly sustain HSC (data not
shown).
Since our cultures sustained HSC surprisingly well, we
continued the cultures another 5 weeks (day 60) corresponding to ;260-fold expansion (;1018-fold) before we
measured again their HSC content. Although HOX cultures
did not any more harbor easily detectable frequencies of
HSC, NUP cultures still contained them at robust levels (at
least 1/200 000) corresponding to ;100-fold reduction from
the original frequency i.e. HSC were expanded ;1016-fold
(Fig. 2, middle panel).
Immortalized bone marrow stem cells
We failed to detect HSC in cultures continued for further
2 weeks (day 74, Fig. 2, lower panel). Possibly, we could
have found some HSC by injecting more cultured cells but it
is clear that cultures gradually lose their genuine HSC despite vigorous factor-dependent growth. Pleasingly though
these extensively cultured cells did not become leukemogenic either (no abnormal expansions in recipient mice) and
hence our genetic manipulation and the long in vitro culture
had not induced or selected such aggressive variants.
1217
did not compromise the ability of NUP cells to differentiate
to functional DC (and to rescue lethally irradiated recipients)
in vivo and that they continued to over-express OVA, PD-L1
and PD-L2 (Fig. 6B). Surprisingly, these DC triggered as efficiently as OVA-only DC the proliferation of naive OVAspecific transgenic T cells although as expected they did not
trigger effector cytokine secretion (H. Khameneh, C. Ruedl, in
preparation). These results underscore the fact that NUP cultures are robust tolerating successive manipulations while
maintaining their potential for differentiation.
Serial transplantation of transduced cells
To further demonstrate the expansion and maintenance of
HSC in our cultures, we have performed serial transplantation experiments (Fig. 4). To challenge the system, we first
cultured the HOXB4-transduced BM cells for 3 weeks i.e. expanded them about million fold and then injected them into
congenic lethally irradiated mice (105 per mouse, without
competitors). After 4 months, the BM cells were isolated
and again cultured, now for 1 week and then injected to secondary recipients (106 per mouse). Again 4 months later, 106
BM cells from the secondary recipients without the culture
were transferred to tertiary hosts and these were then analysed 4 months later for multilineage reconstitution (Fig. 4).
Recipients in every transfer generation were robustly reconstituted giving further support that real HSC were expanded.
Again, we did not detect any gross hematological abnormalities in any of the recipients.
Functional in vitro differentiation of transduced cells
Since the cells from our long-term cultures were able to
functionally repopulate all the lineages in vivo, to rescue lethally irradiated mice and to support T cell dependent antibody responses (data not shown), we wondered if they also
retained their capacity to differentiate in vitro to desired lineages. To do this, we cultured the cells from a 4-week-old
NUP line in the presence of GM-CSF or FLT3L to generate
different types of DCs (myeloid-related and plasmacytoid
DCs) and then compared their efficiency as antigen presenting cells to that of DCs derived similarly from normal BM
(Fig. 5). Encouragingly, NUP cells were able to efficiently
generate both myeloid and plasmacytoid DCs with typical
phenotypes (Fig. 5A). In addition they stimulated T cells as
well as their normal counterparts (Fig. 5B).
Immortalized lines tolerate cumulative and time-consuming
manipulations
To show the resilience of our system for cumulative and successive manipulations, we transduced a 3-week-old NUP
line with a retrovirus coding for OVA-IRES-EGFP and then
selected the green cells by cell sorting. Portion of these cells
were then further transduced with a retrovirus coding for PDL1 and again by cell sorting PD-L1-positive population was
isolated and then further transduced and selected for PD-L2
expression (Fig. 6A). Finally, they and OVA-only expressing
controls were injected in lethally irradiated recipients that
were sacrificed 3 weeks later and spleen DCs were isolated
for in vitro functional assays. As shown in Fig. 6(B), three
successive genetic modifications followed by cell sortings
Discussion
Here, we have extended earlier observations that ectopic expression of HOXB4 or its modified forms can sustain HSC
activity (11–14). In fact, as shown here, it is in practice possible to ‘immortalize’ HSC and their MPPs by interfering their
transcriptional circuitry without necessarily compromising
their physiological function and importantly without inducing
obvious malignant behavior. Therefore, in principal, it is possible to dramatically delay the exhaustion of HSC in culture.
It is certainly worth the effort to try to decipher the relevant
molecular pathways that are induced or enfeebled by the
ectopic expression of HOXB4 and NUP98-HOXB4 (29). The
knowledge of these pathways would allow us not only to develop more specific and less drastic ways to manipulate
HSC but also to understand and ultimately control hematological malignancies.
Since these lines can behave as their normal counterparts
in vitro and in vivo in all aspects tested, they provide us (especially NUP98-HOXB4-transduced BM cells) a practical tool to
study any hematological lineage. These lines are easy to derive from any genetic background and since they sustain their
repopulating properties for long time in culture they make
time consuming procedures like genetic complementation
and cell sorting possible providing high throughput alternative
or preview to more cumbersome germ line manipulations.
Funding
Biomedical Research Council (06/1/22/19/471) to C.R. and
(06/1/22/19/469) Singapore Immunology Network (SiGN-06004) to K.K.
Acknowledgements
Authors wish to thank Ton Rolink for critical reading of the manuscript,
G. Sauvageau and T. Nakamura for plasmids and D. van Essen for
introducing the HOXB4 system in the laboratory. The authors have no
conflicting financial interests.
Abbreviations
BM
DC
GM-CSF
HSC
LSK
MPP
NUP
OVA
PD-L
SCF
bone marrow
dendritic cell
granulocyte macrophage colony-stimulating factor
hematopoietic stem cell
Lin Sca-1+c-Kit+
multipotent primitive progenitor
Nucleoporin 98
ovalbumin
programmed death-1 ligand
stem cell factor
1218 Immortalized bone marrow stem cells
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